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. 2023 Mar 29;616(7956):357–364. doi: 10.1038/s41586-023-05870-7

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 1 — a, Workflow for PVC production in E. coli. The PVC locus was synthesized de novo and cloned into two separate plasmids: a structural/accessory plasmid (pPVC) containing pvc1-16 and a payload/regulatory plasmid (pPayload) containing payloads to be delivered (Pdp1/Pnf) along with several genes thought to regulate PVC gene expression (PAU_RS16570-RS24015). b, Comparison of PVC length distributions from this manuscript and from a previous work⁹. A representative TEM image (with ROIs used to measure PVC lengths highlighted yellow) is also shown. Scale bar, 200 nm. c, HHpred/Pfam annotation of putative PVC regulatory genes (PAU_RS16570-RS24015). At least one gene (PAU_RS16560) contains a predicted domain known to be involved in gene regulation (LysR-type transcriptional regulator domain). d, Bulk protein yields after purification (from 500 mL of bacterial culture) with or without PAU_RS16570-RS24015. A roughly 100-fold increase in protein yield was observed when these genes were included alongside pvc1-16. Values are mean ± s.d. with n = 3 biologically independent reiterations of the purification procedure. e, SDS–PAGE gel depicting the results of purification with or without PAU_RS16570-RS24015, confirming the result from (d). f, TEM images depicting the results of purification with or without PAU_RS16570-RS24015, further confirming the result from (d), and indicating the resulting payload-deficient particles still retain a canonical structure. Scale bar, 200 nm. g, RT-qPCR analysis of PVC expression with or without PAU_RS16570-RS24015. No significant depletion of mRNA levels for pvc1-16 were observed in cells lacking PAU_RS16570-RS24015, indicating these genes do not play a straightforward role as transcriptional regulators for this locus. Values are mean with n = 3 separate qPCR wells for each condition. h, Western blot against a structural PVC component (Pvc12) in the unpurified bacterial culture as well as a purified sample. A Pvc12 band was observed in bacteria lacking PAU_RS16570-RS24015, demonstrating these genes are not necessary for the expression of at least one PVC structural protein. This may suggest PAU_RS16570-RS24015 affect the assembly (rather than expression) of PVC particles.

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